In this grant application we propose to investigate the antifolate activities of several close analogs of the vitamin folic acid which are altered in the pteridine ring system. Specifically, we will replace the: nitrogen atom at the 8th position of folic acid and some of is analogs with a divalent oxygen, and will investiate the potential of these compounds as synthetic substrates of dihydrofolate reductase. in addition, we will investigate the antifolate activities of these 7,8-dihydro-8-oxa analogs using two microorganisms. The enzymatic and catalytic reduction products of these analogs will be tested as potential inhibitors of thymidylate synthetase. All compounds will be submitted to NCI for in vivo antitumor screening. We also intend to carry out structural modifications on folic acid and aminopterin by expanding the 6-membered pyrazine ring to 7-membered ring by interposing a methylene group between carbons 6 and 7. These compounds will have a dihydro pyrimido diazepine ring system instead of a pteridine ring. Both of these compounds, each possessing a 4-hydroxy or a 4-amino group, will be evaluated as a substrate or as an inhibitor of dihydrofolate reductase, respectively. Several close analogs of folic acid possessing a dihydro pyrimido diazepine ring system will be synthesized and evaluated for their antifolate and in vivo antitumor activity.